選殖及表現新穎之人類和斑馬魚第八型17-beta羥基固醇去氫酶

利用生物資訊資料庫NCBI (National Center for Biotechnology Information)中的搜尋比對工具BLAST (Basic Local Alignment Search Tool)，搜尋比對到一組相對應的斑馬魚(編號AL672176/ CAD54662)與人類(編號NM_014234/ NP_055049)羥基固醇去氫酶。此斑馬魚羥基固醇去氫酶cDNA之開放閱讀框架(Open Reading Frame)含有771個鹼基對，可轉譯成256個胺基酸，估計分子量26.970 kDa;與人類已完全被放大出來，而人類羥基固醇去氫酶cDNA之開放閱讀框架含有786個鹼基對，可轉譯成261個胺基酸，估計分子量26.974 kDa。 藉由pQE表達質體系統，將斑馬魚與人類羥基固醇去氫酶表達與純化出來。再以12%SDS-PAGE分析此重組蛋白質，結果發現斑馬魚和人類蛋白質的分子量分別約為32 kDa與29 kDa。 此酵素經西方墨點分析(Western blotting)與北方墨點分析(Northern blotting)，發現在大鼠的腦、心、肺、肝、脾、腎、腸、肌肉、睪丸皆有表現。之後利用酵素動力學分析(enzyme kinetic assay) 測試酵素反應，結果發現此斑馬魚與人類酵素以NAD+為輔酶，以16α- hydroxyestrone為受質，其比活性分別為62.42和311.17 unit/mg。因此，推斷此酵素可能為16α- hydroxysteroid dehydrogenase。

In this study, the search tool BLAST (Basic Local Alignment Search Tool), based on NCBI (National Center for Biotechnology Information) was used against nucleotide and protein databases. The analysis found a hydroxysteroid dehydrogenase (HSD) of zebrafish (NCBI accession numberAL672176/ CAD54662) and human (NCBI accession number NM_014234/ NP_055049). The HSD cDNA of zebrafish and human were successfully amplified with a continuous open reading frame of 771 bps and 786 bps encoding a protein of 256 and 261 amino acids with a calculated molecular mass of 26.970 kDa and 26.974 kDa respectively. Zebrafish and human HSD were cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant proteins have mobility of 32 kDa and 29 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western and Northern analysis showed that HSD is expressed in the rat tissues, such as brain, heart, intestine, kidney, muscle, lung, liver, spleen, and testis. Following expression, purification and the kinetic studies, using 16α hydroxyestrone and NAD+ as substrates, the specific activities of Ni2+ column purified recombinant zebrafish and human HSD were 62.42 and 311.17 unit/mg respectively. Therefore, we renamed these enzymes 16α-hydroxysteroid dehydrogenase.